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SBI4U – Gr.12 University Biology Mr. S. DeBoer Investigation 1.13 – Enzyme Activity Introduction Hydrogen peroxide, H2O2, is a poisonous chemical produced by most cells as a result of normal cell activity. If it accumulates, hydrogen peroxide damages cell components and leads to cell death. Hydrogen peroxide slowly decomposes into harmless water and oxygen gas according to the following equation: 2H2O2 (aq) > 2H2O (l) + O2 (g) (SLOW) Cells produce an enzyme called catalase to speed up this reaction and prevent the accumulation of hydrogen peroxide. catalase 2H2O2 (aq) > 2H2O(l) + O2 (g) (FAST) In this investigation, you will conduct experiments to determine how different environmental conditions affect the speed (rate) at which catalase converts hydrogen peroxide into water and oxygen gas. In this investigation, you will use catalase from liver. Remember that catalase is the enzyme and hydrogen peroxide is the substrate. The speed of the reaction will be determined by measuring the rate at which oxygen gas is produced. A filter-paper disk coated with liver extract containing the enzyme (catalase) is placed into a test tube containing the substrate (hydrogen peroxide solution). As catalase decomposes hydrogen peroxide into water and oxygen gas, bubbles of oxygen collect on the underside of the disk, making it rise to the surface of the hydrogen peroxide solution. The time it takes for the filter-paper disk to rise to the top of the solution will be used as a measure of the rate of enzyme activity. There are four parts to this investigation: • Part 1: Temperature and the Rate of Catalase Activity • Part 2: pH and the Rate of Catalase Activity • Part 3: Enzyme Concentration and the Rate of Catalase Activity • Part 4: Substrate Concentration and the Rate of Catalase Activity The procedure for Parts 1, 2, and 3 will be provided. You will design the procedure for Part 4. Question How do changes in temperature, pH, enzyme concentration, and substrate concentration affect the rate of catalase (enzyme) activity in converting H2O2 (substrate) to water and oxygen? Prediction (please record in your workbooks) (a) Part 1: Predict the effect that a change in temperature will have on the rate of catalase activity. (b) Part 2: Predict the effect that a change in pH will have on the rate of catalase activity. SBI4U – Gr.12 University Biology Mr. S. DeBoer (c) Part 3: Predict the effect that a change in enzyme (catalase) concentration will have on the rate of catalase activity. (d) Part 4: Predict the effect that a change in substrate (hydrogen peroxide) concentration will have on the rate of catalase activity. Materials Catalase (liver extract) 1% hydrogen peroxide (H2O2) solution 3% hydrogen peroxide (H2O2) solution pH 2 buffer solution pH 4 buffer solution pH 6 buffer solution pH 8 buffer solution pH 10 buffer solution Stopwatch/timer 10-mL graduated cylinder Distilled water squirt bottle Hot plate, Ice Metric ruler 3 large beakers for water baths Tape + Pen (for labelling) Lab coat/apron 6 test tubes, test-tube rack Hole-punched filter paper disks Tweezers/forceps Procedure General Procedure for ALL parts 1. Using forceps or tweezers, dip a filter-paper disk into a test tube containing liver extract (catalase enzyme). 2. After removing the filter paper from the liver extract, place it carefully into the test tube containing hydrogen peroxide solution. The disk must be placed just below the surface of the solution so that it sinks to the bottom of the test tube when released. 3. Start timing the moment the disk is released. The disk will fall to the bottom (sometimes it won’t even make it to the bottom) and then begin to rise up again when O2 bubbles begin to form on the surface of the disks. Stop timing when the disk reaches the surface of the solution. (e) Record the times in your workbooks as they are recorded. It is important to remember that the faster the time recorded, the higher the enzyme activity. This can be shown by converting the time into a rate by doing ( 1 time rate ) Part 1: Temperature and the Rate of Catalase Activity 4. The following five water baths are needed for this part: an ice bath (0C), a roomtemperature water bath (20C), a body-temperature water bath (37C), a 45C water bath, and a boiling water bath (100C). Use tap water in a beaker for the room temperature water bath. The 37C and 45C baths are already prepared for you. You will need to prepare the ice bath and the boiling water bath on your own. SBI4U – Gr.12 University Biology Mr. S. DeBoer 5. Using tape and a pen, label five clean test tubes: 0C, 20C, 37C, 45C, and 100C. 6. Place 5 mL of stock liver extract, provided at the side bench, into each of the five labelled test tubes, and immerse each test tube in its appropriate water bath. Let the test tubes incubate for approx. five minutes. 7. Take another clean test tube, and fill it up with approx. 6 cm of 1% Hydrogen Peroxide solution. DO NOT place this test tube in any of the water baths. Heating H2O2 (aq) is dangerous! 8. Using the technique described in steps #1-3, measure the rate of enzyme activity for the catalase at each temperature. 9. When finished and your data is recorded, wash and dry your test tubes for use in Part 2. Part 2: pH and the Rate of Catalase Activity 10. Label five clean test tubes: pH 2, pH 4, pH 6, pH 8, and pH 10. Using your graduated cylinder, measure 5 mL of stock liver extract into each of the test tubes. 11. Rinse your graduated cylinder, and then use it to place 5 mL of each buffer into its appropriately labelled test tube. So, 5 mL of pH 2 buffer into the test tube labelled pH 2, etc. Be sure to rinse your graduated cylinder between each buffer to avoid contamination. 12. Take another clean, empty test tube, just as before, and fill it up with approx. 6 cm of 1% Hydrogen Peroxide solution. 13. Using the technique described in steps #1-3, measure the rate of enzyme activity for the catalase at each pH. 14. When finished and your data is recorded, wash and dry your test tubes for use in Part 3. Part 3: Enzyme Concentration and the Rate of Catalase Activity 15. Label five clean test tubes: 100% catalase, 80% catalase, 50% catalase, 20% catalase, and 0% catalase. 16. Place the following mixtures into each of the test tubes prepared in step 15: a. 100% catalase: 10 mL of stock liver extract only b. 80% catalase: 8 mL of stock liver extract + 2 mL of distilled water c. 50% catalase: 5 mL of stock liver extract + 5 mL of distilled water d. 20% catalase: 2 mL of stock liver extract + 8 mL of distilled water e. 0% catalase: 10 mL of distilled water only SBI4U – Gr.12 University Biology Mr. S. DeBoer 17. Take another clean, empty test tube, just as before, and fill it up with approx. 6 cm of 1% Hydrogen Peroxide solution. 18. Using the technique described in steps #1-3, measure the rate of enzyme activity for the catalase at each enzyme concentration. 19. When finished and your data is recorded, wash and dry your test tubes for use in Part 4. Part 4: Substrate (H2O2) Concentration and the Rate of Catalase Activity (f) Design a procedure to determine the effect that changes in substrate concentration have on the rate of catalase activity, using the same idea as Parts 1-3. Write a list of the materials you will need and the steps you will follow, and, upon approval from your teacher, perform your experiment and record your results in your workbook. Analysis and Evaluation Questions Please answer all questions, (g) – (l), in your workbook. Then begin writing your formal lab report for this lab, including everything discussed by your teacher. The due date will be decided together as a class. Analysis Questions (include answers to these questions in Analysis/Results section) (g) Summarize your results from tables (in Observations) by sketching suitable graphs for Parts 1,2,3, and 4. (h) Analyze all of your results for trends and patterns. (i) Answer the Question at the beginning of the investigation. Evaluation Questions (include answers to these questions in Discussion section) (j) Evaluate your predictions, taking into account possible sources of error. Draw reasonable conclusions (which will also be summarized in your conclusion section). (k) Describe how you could improve your experimental methods and the rate technique. (l) Suggest other experiments you could perform to extend your knowledge of enzyme activity. Due Date:
